PREPARATION AND CHARACTERIZATION OF
HAP COATED- CHITOSAN-ALGINATE PEC
POROUS SCAFFOLD FOR BONE TISSUE
ENGINEERING
A THESIS SUBMITTED IN PARTIAL FULFILMENT
OF THE REQUIREMENT FOR THE DEGREE OF
Master of technology
In
Biotechnology
By
Trupti Umakant Patil
213BM2033
Under the supervision of
Prof. Amit Biswas
Department of Biotechnology & Medical Engineering
National Institute of technology, Rourkela
Orissa, 769008
National Institute of Technology, Rourkela
CERTIFICATE
This is to certify that thesis entitled “Preparation and characterization of HAp coated
Chitosan-Alginate PEC porous scaffold for bone tissue engineering” by Miss Trupti Patil,
submitted to the National Institute of Technology, Rourkela for the Degree of Master of
Technology is a record of bonafide research work, carried out by her in the Department of
Biotechnology and Medical Engineering under my supervision and guidance. To the best of my
knowledge, the matter embodied in the thesis has not been submitted to any other University/
Institute for the award of any Degree or Diploma.
Dr. (Prof.) Amit Biswas
Assistant Professor
Department of Biotechnology & Medical engineering
National Institute of Technology,
Rourkela, Orissa, 769008
Acknowledgment
I feel immense pleasure and privilege to express my deep sense of gratitude, indebtedness and
thankfulness towards all those people who have helped, inspired and encouraged me during the
preparation of this report.
I would like to thank my guide Dr. Amit Biswas, who provided me this opportunity to highlight
the key aspects of an upcoming technology and guided me during the project work preparation, I
would also like to thank Prof. Krishna Pramanik head of department (BM), Prof. S.K.Pratihar
(CR) and Mr. L.K. Mohanty (CR) for their support and coordination.
I am also taking the opportunity to thank other faculty members and supporting staff members of
the Department of Biotechnology and Medical Engineering for their timely cooperation and
support at various phases of experimental work. I would like to thank Prof. Indranil Banerjee and
Prof. Sirsendu Ray for always supporting me and nurturing me.
I would like to thank whole heartedly my parents, family members whose love and unconditional
support, both on academic and personal front, enabled me to see the light of this day.
Last but not the least, I would also like to thank my seniors Ms. Varshini, Sahely, Senthilguru,
Bhisham Singh and all my batchmates, specially Gaurav, Rohan, Anupriya, Vinay, Joseph ,
Goutham, Gauresh and Usha for their kind support and helping behaviour.
Thanking you,
Trupti Umakant Patil
213BM2033
Dept. of Biotechnology and Medical Engineering
National Institute of Technology, Rourkela
Table of Contents
List of figures I
Nomenclature II
Abbreviations III
Abstract IV
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW
2.1 Bone tissue engineering……………………………………………………………...4.
2.2 Biodegradable scaffolds……………………………………………………………..5
2.2.1 Polymeric scaffolds
2.2.2 Hybrid scaffolds
2.3 Chitosan and alginate as ideal biomaterials for bone tissue engineering…………..7
2.4 Chitosan-alginate polyelectrolyte complex (CAPEC)………………………………8
2.5 Preparation of CAPEC hybrid scaffold……………………………………………..9
2.5.1 Chitosan-alginate porous scaffolds
2.5.2 Chitosan-alginate fibrous scaffolds
2.6 HAp derived inorganic coatings……………………………………………………12
2.7 Preparation of nano sized HAp……………………………………………………...13
CHAPTER 3: AIMS & OBJECTIVES 14
CHAPTER 4: MATERIALS & METHODS
4.1 Materials……………………………………………………………………………..15
4.1.1 Preparation of scaffold
4.1.2 Synthesis of hydroxyapatite
4.2 Methods…………………………………………………………………………...15-17
4.2.1 Preparation of CAPEC scaffold
4.2.2 Synthesis of hydroxyapatite (HAp)
4.2.3 Preparation of HAp coated-CAPEC scaffold (CAPEC/HAp)
4.3 Characterization…………………………………………………………………..17-20
4.3.1 Average particle size
4.3.2 Morphology
4.3.3 Porosity
4.3.4 Phase analysis
4.3.5 Functional analysis
4.3.6 Mechanical strength
4.3.7 Swelling behavior
4.3.8 In-vitro biodegradation study
CHAPTER 5: RESULTS & DISCUSSION 21-29
5.1 Average Particle size………………………………………………………..21
5.2 Preparation of scaffolds……………………………………………………..22
5.3 Morphology & pore size……………………………………………………..22
5.4 Porosity………………………………………………………………………23
5.5 Phase analysis………………………………………………………………..24
5.6 Functional analysis…………………………………………………………..25
5.7 Mechanical strength………………………………………………………….26
5.8 Swelling behavior……………………………………………………………27
5.9 In-vitro biodegradation……………………………………………………....28
CHAPTER 6: SUMMARY & CONCLUSION 30
SUGGESTED FUTURE WORK 31
REFERENCES 32-33
I
List of figures
I. Figure 4.2.1a Flowchart for preparation of CAPEC scaffolds
II. Figure 4.2.2a Schematic representation of methodology for synthesis of HAp
III. Figure 5.1.1 Size distribution profile of HAp sample
IV. Figure 5.2.1 a) Developed CAPEC scaffold, b) CAPEC/HAp1 scaffold, c) CAPEC/HAp2
scaffold
V. Figure 5.3.1 SEM images of CAPEC scaffold (a & b) , CAPEC/HAp1 scaffold (c & d)
and CAPEC/HAp2 scaffold (e & f)
VI. Figure 5.5.1 XRD spectra of CS-AG, CAPEC scaffold, HAp powder and CAPEC/HAp1
Scaffold
VII. Figure 5.6.1 FTIR spectra of a) pure chitosan, b) pure alginate, c) CAPEC/HAp1 scaffold
and d) HAp powder.
VIII. Figure 5.7.1 Compressive strength (MPa) of CS-AG, CAPEC, CAPEC/HAp1 and
CAPEC/HAp2 Scaffolds
IX. Figure 5.8.1 Images of CAPEC (a), CAPEC/HAp1 (b) and CAPEC/HAp2 (C) scaffolds
after two days of immersion in PBS.
X. Figure 5.8.2 Percentage swelling of scaffolds as a function of sample immersion time in
PBS: CAPEC, CAPEC/HAp, CAPEC/HAp2 Scaffolds
XI. Figure 5.9.1 Percentage weight remaining of scaffolds as a function of sample immersion
time in PBS: CAPEC, CAPEC/HAp, CAPEC/HAp2 Scaffolds
II
Nomenclature
I. BTE Bone tissue engineering
II. CS Chitosan
III. AG Alginate
IV. PEC Polyelectrolyte complex
V. CAPEC Chitosan-alginate polyelectrolyte complex
VI. HAp Hydroxyapatite
VII. CAPEC/HAp HAp coated-Chitosan-alginate polyelectrolyte complex
III
Abbreviations
XRD X-ray Diffraction
DLS Dynamic Light Scattering
FTIR Fourier Transform Infrared Spectroscopy
SEM Scanning Electron Microscopy
PBS Phosphate Buffered Saline
IV
ABSTRACT
This thesis reports the development of 3D porous hybrid scaffold using chitosan-alginate
polyelectrolyte complex (CAPEC) by freeze drying method. The CaCl2 cross-linked CAPEC
scaffold was further coated with synthesized HAp by dip coating technique. The HAp synthesis
was done by wet chemical precipitation method in organic solvent i.e. ethanol. The average
particle size of synthesized HAp was found to be ~159 nm. The CAPEC and HAp coated-
CAPEC (CAPEC/HAp) scaffolds were assessed for their porosity, pore size, morphology,
mechanical strength, swelling behavior, in-vitro biodegradation. The pore size and morphology
of developed scaffolds was studied by scanning electron microscopy (SEM) & no apparent
change in pore size was found in case of CAPEC/HAp. Both the scaffolds possessed desired pore
size with interconnected pore network. Whereas, the porosity was decreased with the increase in
HAp coating, but it is still high enough for bone tissue engineering application. The mechanical
strength of CAPEC scaffold increased with the coating of HAp, compressive strength was
increased from 0.469 MPa to 0.61 & 0.67 MPa. The CAPEC/HAp scaffolds also exhibited
favorable swelling behavior and biodegradation. Overall, the study has demonstrated that for
non-load bearing bone tissue engineering applications, the developed CAPEC/HAp scaffolds do
have the potential use.
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1. INTRODUCTION
Bone is a dynamic and highly vascularized tissue which forms the foundation of our bodily
locomotion[1]. The main role of bone is to provide structural support for the body. It also
provides protection for our internal organs and load bearing capacity to skeleton[2]. Hence,
Trauma, Injury or bone related diseases causing impairment or loss of bone tissue leads to
reduced quality of life in many patients[3]. Growing knowledge of bone related abnormalities
has permitted diagnosis capabilities and therapeutic solutions[4] which involve utilization of
bone grafts to escalate bone repair and regeneration. Whereas, these treatments are associated
with various issues such as donor site morbidity, limited availability, immune rejection, and
pathogen transfer[1]. Failure rates in these techniques has prompted a lot of research interest
among the scientific community for an alternative solution[5]. It is in this context that Bone
Tissue Engineering (BTE) has emerged as an alternative strategy to treat bone abnormalities
through the development of a biologically active substitute so called tissue engineered
scaffold[6]. BTE typically employs the coordinated manipulation of cells, biodegradable,
biomimetic scaffolds and biologically active signaling molecules[7]. A biodegradable scaffold is
a 3D matrix which is inserted into the site of defect or lost bone. It supports and encourage bone
tissue regeneration while it gradually degrades and is replaced by new bone tissue[8]. For
success of BTE, scaffolds should have optimum porosity, appropriate pore size and
interconnected pores for the passaging cells, nutrients, metabolites and signal molecules. Also,
the scaffold should be nonimmunogenic, nontoxic, biodegradable at ideal rate corresponding to
the rate of new tissue formation and biocompatible [9]. Besides, it should be structurally stable
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and capable of providing desired mechanical strength and temporary mechanical integrity.
Furthermore, scaffold must facilitate bone formation by stimulating cell adhesion, proliferation
and regulating osteogenic differentiation of host cells [3, 10, 11]. In this context, material
properties and appropriate fabrication technique are prerequisites for the development of 3D
scaffold for bone tissue regeneration.
For BTE scaffolds, both polymers and bioactive ceramics have been developed and analyzed.
Chemical composition of bioactive ceramics resembles the natural bone composition; they also
promote osteogenesis and are capable of making bonds with host bone. But, clinical use of
bioceramics is limited due to their brittleness and low biodegradation rate. On the other hand,
biopolymers have some distinct advantages over bioceramics. Biopolymers display mechanical
properties compatible with human cancellous bone. They can be fabricated easily into desired
shapes and can be modified to certain extent to optimize their biodegradation rate and
mechanical properties for specific applications [1, 8]. Even so, biopolymeric scaffold fail to
retain their shape in long term. Development of an interconnected bioceramic-biopolymer
scaffold takes advantages of both the components to meet mechanical and physiological
requirements of the host tissue[12].
Over the last two decades chitosan being a natural polymer has played major role in bone tissue
engineering. It is also known as best bioactive material, as it is biodegradable, bioactive and
osteoconductive [11]. Next to chitosan, alginate, another natural polysaccharide is the most
widely used biomaterial in the field of bone tissue engineering [13]. Although, Chitosan and
alginate are generally accepted biomaterials, due to their low mechanical strength and
incompetency of retaining shape for long period of time, they somehow fail as ultimate
biomaterials for bone tissue engineering [8]. Being oppositely charged, chitosan spontaneously
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associates with alginates in solution to form polyelectrolyte complexes (PECs) upon mixing [14].
3D porous scaffold can be fabricated using chitosan- alginate PECs using freeze drying
technique [8]. Scaffolds developed from chitosan-alginate PECs show significant improvement
in mechanical and biological properties when compared to its counterparts[8]. Hence they are
assumed to be having potential for bone tissue engineering purpose.
To further improve the mechanical and biological properties of scaffold and facilitate
osseoconduction, biologic coating can be used. Hydroxyapatite (HAp) being chemically similar
to the apatite of human bone, provide osteoconductive approach augments new bone tissue
formation [15]. A very simple, energy and time saving technique, dip coating can be used for
coating HAp on chitosan-alginate PEC scaffold.
In this strategy, in the present work chitosan-alginate PEC scaffolds coated with HAp will be
fabricated by applying freeze drying and dip coating method. The developed scaffolds will then
be characterized to study their potential for bone tissue engineering.
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2. LITERATURE REVIEW
2.1 Bone tissue engineering Bone is a dynamic and highly vascularized tissue which forms the foundation of our bodily
locomotion[1]. The main role of bone is to provide structural support for the body. It also
provides protection for our internal organs and load bearing capacity to skeleton[2]. Hence,
Trauma, Injury or bone related diseases causing impairment or loss of bone tissue leads to
reduced quality of life in many patients[3]. Growing knowledge of bone related abnormalities
has permitted diagnosis capabilities and therapeutic solutions[4] which involve utilization of
bone grafts to stimulate bone repair and regeneration. Currently, over 400,000 and 600,000 bone
grafting operations are performed in Europe and America, respectively and the need of bone
grafts is increasing[16]. Clinical practices have shown that autografts being non-immunogenic
and histocompatible, serve as an excellent bone graft[1].Whereas, these treatments are associated
with major concerns such as donor site morbidity, limited availability. Allografts and xenografts
may raise other issues such as immune rejection, and pathogen transfer[1]. Increasing need of
bone grafts has also resulted in developing bone implants. Traditional porous bone implants are
made of ceramics and metals. But these implants are unable to integrate properly with the host
tissue, resulting in poor surgical outcome. Other reasons for unsatisfactory outcomes involve
mechanical mismatch, corrosion and wear[17]. Failure rates in these techniques has prompted a
lot of research interest among the scientific community for an alternative solution[5]. It is in this
context that Bone Tissue Engineering (BTE) has emerged as an alternative strategy to treat bone
abnormalities through the development of a biologically active substitute so called tissue
Page | 5
engineered scaffold[6]. BTE typically employs the coordinated manipulation of cells,
biodegradable, biomimetic scaffolds and biologically active signaling molecules[7].
2.2 Biodegradable scaffolds
A biodegradable scaffold serves as a temporary skeleton in bone tissue engineering. It is
implanted into the defect site in order to support and encourage bone tissue regeneration. As the
new bone tissue is formed, a biodegradable scaffold gradually degrades being replaced by newly
formed tissue [1]. A scaffold must provide a porous network for the transport of nutrients, signal
molecules, metabolite and cells. It should be biocompatible and rate of biodegradation should be
equal to the rate of new tissue formation[9]. Besides, it should be structurally stable and capable
of providing desired mechanical strength and temporary mechanical integrity. Furthermore,
scaffold must facilitate bone formation by stimulating cell adhesion, proliferation and regulating
osteogenic differentiation of host cells [3, 10, 11].
Following are the requirements of an ideal scaffold in bone tissue engineering,
I. Biocompatibility:
A scaffold should be nontoxic and nonimmunogenic to the host tissue and it should be
able to support normal cellular activities, cell signaling. It should allow cell adhesion,
proliferation and formation of extracellular matrix on its framework. In addition to being
osteoconductive, an ideal scaffold should also be osteoinductive i.e. it should be able to
induce new bone tissue formation.
II. Mechanical Properties:
An ideal scaffold must have mechanical properties equivalent to host bone properties. As
bone is a very complex tissue with a complex biomechanical system, its mechanical
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properties vary widely from cancellous to cortical bone. Compressive strength and
young’s modulus of cortical bone is between 100-200 MPa and 15-20 GPa, respectively.
Whereas in case of cancellous bone it varies from 2-20 Mpa and 0.1-2 GPa. It is difficult
to design an ideal bone scaffold due to this large variation in the mechanical properties of
bone tissue.
III. Pore size:
For bone tissue engineering, an ideal scaffold should have pore size of at least 100µm.
However, scaffolds with pore size range of 200-350µm are found to be optimum for bone
tissue engineering. Micropores are essential for diffusion of nutrients, cell signaling
molecules whereas macro pores allow tissue ingrowth. It has been reported that scaffolds
with both micro and macro pores perform better than only macroporous scaffold.
Unfortunately, mechanical properties reduce with the increase in the porosity.
IV. Bioresorbability:
An ideal scaffold should be able to degrade with time in-vivo at the same rate as new
tissue formation. It should be resorbed at a controlled rate, creating space for new bone
tissue growth. The degradation behavior of the scaffold varies depending on the
application (site of implant).
Hence, key challenge in bone tissue engineering is to design and manufacture a porous scaffold
with ideal composition, mechanical properties and biodegradability.
2.2.1 Polymeric scaffolds
Biopolymers are known to be bioactive, biocompatible and biodegradable. Polymers can be
processed and tailored to get porous or fibrous scaffolds. Natural polymers such as Alginate,
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chitosan, silk, collagen, hyaluronic acid and fibrin are commonly used in bone tissue
engineering. Poly-lactic acid, poly-glycolic acid and poly-caprolactone are some of the synthetic
polymers used [12].
2.2.2 Hybrid scaffolds
It is well known that any biomaterial cannot satisfy the requirements of bone scaffolds. Hence,
efforts have been made in developing hybrid scaffolds. Hybrid scaffolds can be formed by
combining two or more biomaterials. This allows designing a scaffold with enhanced properties.
The scaffold can be made up of polymer-polymer blend or polymer-ceramic composite.
Polymer-polymer blends can be formed by mixing two or more polymers forming a miscible
blend with enhanced properties. It is reported that chitosan-alginate[8], chitosan-pectin-
alginate[18] blends have been used to design scaffolds for bone tissue engineering. Polymer-
ceramic composites are prepared by mixing a polymer with an inorganic ceramic material.
Composites of hydroxyapatite with polymers such as chitosan[19], alginate[13], PLA, gelatin,
collagen[1] have been reported to be successful in bone tissue engineering.
2.3 Chitosan and alginate as ideal biopolymer for tissue engineering
Chitosan is a natural biopolymer found in cell walls of fungi and shells of marine crustaceans. It
is a copolymer of β-(1→4)-2-acetamido-d-glucose and β-(1→4)-2-amino-D-glucose unit
linkages. Chitosan is considered to be having excellent biocompatibility and bioactivity[8]. it
also has an intrinsic antibacterial property. Chitosan possesses hydrophilic surface which helps in
cell adhesion, proliferation, and differentiation. It has also shown to promote bone tissue growth
and mineral deposition by osteoblast culture. Chitosan is highly capable of forming porous
structure through lyophilization. It can also be tailored into gel , beads, sponges, fibers[19]. In
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spite of its general acceptance as a tissue biocompatible material, chitosan is mechanically weak
and unstable, and unable to maintain a predefined shape for transplantation as a result of
swelling[8]. Hence to overcome the limitations of pure chitosan scaffolds and to develop
scaffolds with enhanced properties, chitosan is combined with other polymers such as alginate,
hyaluronic acid, PLA or bioceramic like hydroxyapatite, calcium phosphate[11].
Next to chitosan, Alginate is the most widely used natural biopolymer in bone tissue engineering.
It is obtained typically from brown seaweed including L. hyperborea, L. digitata, L. japonica,
Macrocystis pyrifera, and Ascophyllum nodosum. Due to alginate’s excellent biocompatibility,
nontoxicity, relative low cost it has found many biomedical applications [20]. Alginate is widely
used as an instant gel for bone tissue engineering [2]. Also, it can be easily modified to form
hydrogels, sponges, foams, microspheres, and fibers [13]. Alginate gels can be used to deliver
osteoinductive factors, bone forming cells or both for bone regeneration[20]. However, alginate
gels do not possess sufficient mechanical strength for load bearing applications. Hydroxyapatite
can be added to alginate gels to improve the mechanical properties as well as to enhance bone
tissue formation[13].
2.4 Chitosan-alginate polyelectrolyte complex (CAPEC)
Although, Chitosan and alginate are generally accepted biomaterials for bone tissue engineering,
they possess low mechanical strength and they are incompetent of retaining shape for long period
of time. To combine their individual properties, Chitosan-alginate composites are developed.
This composite is one of the most studied materials in bone tissue repair.
Chitosan is positively charged at low pH values (below its pKa value), whereas alginate is an
anionic polymer. Being oppositely charged, when mixed with each other they spontaneously
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associate to form polyelectrolyte complexes [3]. The polyelectrolyte complex (PEC) is formed
through ionic interaction between protonated amines on chitosan and carboxylate moieties on
alginate. PECs can be formed in the form of membrane [21], capsule [22], fiber [23], and
scaffold [24].
The formation and stability of these PECs depend on many factors such as the degree of
ionization of chitosan and alginate, charge density, the charge distribution over the polymeric
chains, polymer concentration, their mixing order, the mixing ratio, the duration of the
interaction, molecular weight of the polyelectrolytes as well as the temperature, ionic strength
and pH of the reaction medium [3]. Biodegradation studies on PECs have showed the effect of
lysozyme on the PEC is negligible. PECs have a high ability of lysozyme adsorption, but due to
strong interaction between chitosan and alginate polymeric chain enzymatic hydrolysis is
hindered[21]. The rate of biodegradation may be regulated by changing the polymer ratio, it
indicates that PECs has high potential for scaffolds in tissue engineering.
It is also reported that scaffolds developed from CAPECs have higher mechanical strength than
the individual polymer scaffolds. CAPECs can be fabricated into a scaffold with very high
porosities and high interconnectivity. Also crosslinking with calcium ions provide more rigidity
to the scaffold, this allows the scaffold to absorb solution without considerable swelling[22].
Thus scaffolds developed from chitosan-alginate PECs are assumed to be having potential for
bone tissue engineering purpose.
2.5 Preparation of CAPEC hybrid Scaffold
Using Chitosan-alginate, porous scaffold as well as fibrous scaffolds can be fabricated. Porous
scaffold can be prepared through a simple lyophilization technique [8, 9, 18, 23, 24]whereas
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preparation of fibrous scaffold involves various techniques like wet spinning, spray spinning to
produce the PEC fibers and then lyophilization of these fibers to get 3D scaffold[25, 26].
2.5.1 Chitosan-alginate porous scaffolds
Chitosan-alginate porous scaffolds can be fabricated by various methods. A general method
involves gradual mixing of the two components to form PECs and using these PECs, a scaffold
is fabricated. Other methods involve fabrication of a framework of either component followed by
immerging it into the other components solution to facilitate the formation of PEC. These
methods can be described as follows:
I. Scaffold fabrication using PECs formed by gradual mixing of Chitosan and Alginate,
II. Immersion of Alginate scaffold in Chitosan solution leading to PEC formation (Alginate-
chitosan PEC Scaffold),
III. Immersion of Chitosan scaffold in Alginate solution leading to PEC formation (Chitosan-
alginate PEC Scaffold).
Scaffold fabrication using PECs
This method is involves the formation of PECs first, followed by fabrication of scaffold by
freeze drying the PECs. PEC formation is achieved by blending the chitosan and alginate
solutions together. Order of addition of one polymer into other, blending conditions, pH of the
solution, the ionic strength and temperature affect the size of PECs formed. By optimizing these
conditions PECs of nanometer to micrometer range can be produced. To increase the mechanical
strength of the scaffold made, it is cross-linked using Calcium chloride solution. This results in
the crosslinking of the alginates present in PECs enhancing the structural stability and
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mechanical strength. After crosslinking, the scaffold is freezed again and lyophilized. It has been
reported that scaffold fabricated with this technique has significantly improved mechanical
strength than its chitosan counterpart. Also ~92% porosity and a compressive modulus of
8.16MPa and yield strength of 0.46 MPa, respectively can be achieved. The scaffolds were found
to be osteoconductive [8].
Alginate-Chitosan PEC scaffold
Pure alginate scaffold is immersed in chitosan solution to allow on site PEC formation. This
immersion is then lyophilized to get alginate-chitosan PEC scaffold with interconnected pores.
To further increase the mechanical strength and structural stability of the scaffold, it is
crosslinked using calcium chloride. It is reported that, The AG–CS PEC scaffold fabricated
through above method exhibites higher compressive strength compared with pure alginate
scaffold[23].
Chitosan-Alginate PEC scaffold
In this method chitosan scaffold is formed first and then it is immersed in alginate solution
facilitating the formation of PEC. After dipping the scaffold in alginate, it is freezed and
subsequently lyophilized. This method allows uniform PEC formation without destroying fine
pore structure of the scaffold. It is also shown to be activating the production of mineralized
matrix from the cells [24].
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2.5.2 Chitosan-Alginate Fibrous scaffold
Chitosan-Alginate fibrous scaffold can be fabricated by extruding one polymer solution from a
nozzle into other polymer solution to formulate fibers. These fibers are then freeze dried to get
the scaffold.
In spray-spinning, alginate solution is agitated continuously using magnetic stirrer while chitosan
solution is being sprayed into it. Because of the agitation the sprayed chitosan solution is sheared
into streamline. This results in the formation of elongated fibers of chitosan-alginate PEC. The
spray-spun fibers thus formed are then centrifuged and washed. The collected fibers are
fabricated into a 3D scaffolds by freeze drying them[26]. Spray spun fibrous scaffold has been
reported to be investigated for its potential use as connective tissue regeneration. But it can be
optimized further for applications of BTE.
In wet spinning, alginate fibers are produced first with the help of calcium chloride crosslinker.
These alginate fibers are then immersed in the chitosan solution. The chitosan coating results in
the formation of an alginate-chitosan PEC. These chitosan-alginate PEC fibers are then freeze
dried for further use [25]. Wet spun fibrous scaffolds also need to be optimized for their
application in BTE, due to lower their degradation rate and increase mechanical strength.
2.6 HAp-derived inorganic coating
A natural polymer-based, highly porous scaffold with better mechanical strength and biological
property is yet to be developed. In order to achieve this various surface techniques have been
employed to produce different types of coatings on the surface of scaffolds[16]. Hydroxyapatite
(HAp) being chemically similar to the apatite of human bone, HAp coating can be used to
improve biologic function of the scaffold. Also, HAp is capable of forming tight bonds with the
Page | 13
host bone, it also facilitates cell adhesion, proliferation and differentiation. Conventionally, HAp
is thought to be osteoconductive but reports have shown that with certain 3 dimensional
geometries it is able to bind with endogenous bone morphogenic proteins and designated to be
having osteoinductive properties[15]. It also improves the mechanical strength of the scaffold as
a result of its crystal structure [27].
HA coatings can be produced by dip coating, spin coating or alternate soaking method, etc. Dip
coating method involves immersion of scaffold in the HAp dispersion at a specific rate and
withdrawal of the scaffold followed by evaporation of the solvent. Optimum thickness HAp
coating can be achieved by repetitive cycles of dip coating [28].
Alternate soaking of scaffold in calcium chloride solution and disodium hydrogen phosphate
solution also produce HAp coating. But size of HAp produced through this method ranges in
micrometers. This results in significant decrease of the porosity [29]. Also, alternate soaking
method is easy but time consuming method, whereas dip coating seems to be simple and quick
method.
2.7 Synthesis of nano-sized HAp
There are several methods of preparing nano HAp reported in the literature, including wet
chemical precipitation, biomimetic deposition, sol-gel and electrodeposition [30]. Wet chemical
precipitation generally involves use of water as a solvent to mix calcium hydroxide and
phosphoric acid to form HAp. Diammonium hydrogen phosphate and calcium nitrate have also
been used to produce HAp through wet chemical precipitation. It has b7een reported that The
rod-like shape HAP crystals with a diameter of ~ 6nm and length of 75 nm formed at room
temperature in ethanol solvent[31].
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3. AIMS & OBJECTIVES
The aim of this study is to develop a 3D porous hybrid scaffold using chitosan-alginate
polyelectrolyte complex for bone tissue engineering applications & to modify the developed
scaffold in order to achieve desired mechanical strength and bioactivity.
The specific objectives of this study are:
1. To develop a porous scaffold from chitosan-alginate polyelectrolyte complex (CAPEC),
2. To synthesize hydroxyapatite (HAp),
3. To coat the developed CAPEC scaffold with HAp in order to improve its mechanical
strength and bioactivity,
4. To characterize the HAp coated CAPEC scaffold (CAPEC/HAp) for its pore size,
porosity, mechanical strength, swelling behavior, in-vitro biodegradation,
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4. MATERIALS & METHODS
4.1 Materials
4.1.1 Preparation of CAPEC scaffold
Chitosan (From Shrimp Shells, Degree of deacetylation >= 75%), Sodium alginate, Sodium
hydroxide pellets, Acetic acid, Calcium chloride were purchased from Himedia, India. Sodium
tripolyphospate was procured from Sigma Aldrich (USA).
4.1.2 Synthesis of Hydroxyapatite (HAp)
Commercially available Diammonium hydrogen phosphate ((NH4)2HPO4) (Rankem), calcium
nitrate tetrahydrate (Ca(NO3)2.4H2O) (Loba Chemie pvt. Ltd.), ammonia solution (NH4OH)
(Rankem), and absolute (anhydrous) ethanol (Sigma-Aldrich) were purchased.
4.2 Methods
4.2.1 Preparation of CAPEC scaffold
CAPEC scaffolds were prepared by the method reported elsewhere [8] with some minor changes.
Figure 4.2.1a shows the flow chart for preparation of CAPEC scaffold. Briefly, Chitosan
solution was prepared by dissolving chitosan powder (4.8 g) in 80 ml 1N acetic acid. 4.8 g of
sodium alginate was dissolved in 120 ml 1N NaOH. The two solutions were mixed together
using an overhead stirrer at 4000 rpm, under constant stirring. The homogenous chitosan-alginate
(4.8% w/v) solution obtained contains 2.4% each chitosan and alginate. The CS-AG solution was
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further homogenized at 4000 rpm for 15 minutes [21]. The diameter of dispersing element used
was 25 mm. 2N acetic acid was used to adjust the pH of CS-AG solution to physiological pH
(pH 7.4). The CS-AG solution was introduced into 6-well culture plates and maintained at -200C
for 24 h. The samples were then freeze dried at -550C for 48 h. 1% CaCl2 was used to crosslink
the freeze dried scaffolds for 10-15 minutes. The scaffolds were washed thoroughly by distilled
water to remove traces of unbound cross linker. The samples were then freeze dried.
Figure 4.2.1a Flow chart of preparation of CAPEC scaffold
Chitosan solution
(4.8 g in 80 ml 1N Acetic acid)
Alginate solution
(4.8 g in 120 ml of 1N NaOH)
Blending using overhead stirrer
(3200 rpm)
Homogenization
4000 rpm, 15 min
Freezing at -200C
Lyophilization
At -550
C, for 48 hours
Crosslinking
Using 1% CaCl2
pH 7.4
Lyophilization
At -550
C, for 48 hours
Page | 17
4.2.2 Synthesis of hydroxyapatite (HAp)
Hydroxyapatite was synthesized using wet chemical precipitation method as follows [31]. First,
100 ml of 0.5 M (NH4)2HPO4 & 0.3 M Ca(NO3)2.4H2O were prepared in ethanol. Before
preparing the solution, each reactant was first dissolved in 5 ml of water followed by adjusting
the pH between 10-12 using ammonia solution. Then the solutions were diluted with ethanol to
make up the volume. 0.5 M (NH4)2HPO4 solution was added to 0.3 M Ca(NO3)2.4H2O solution
under constant stirring. The reaction was carried out at 400C for 24 h. The solution was then
filtered and washed, and then precipitate was collected and kept overnight in hot air oven at 400C
for drying.
Figure 4.2.2a Schematic representation of methodology for synthesis of HAp
0.5 M (NH4)2HPO
4
solvent solution
0.3 M Ca(NO3)2.4H
2O
solvent solution pH (10-12)
Rapid mixing
(Constant stirring, 24 hr,
400
C)
Filtration & washing
Collection of
filtrate
Drying at 400
C (12 hr)
HAp powder
Page | 18
4.2.3 Preparation of HAp coated-CAPEC scaffold (CAPEC/HAp)
HAp was deposited on CAPEC scaffold by dip coating method as described in literature [15].
The HAp suspension was prepared by dissolving 0.1 g HAp powder in 20 ml ethanol at room
temperature. The suspension was maintained using ultrasonication bath for 1 hr. Then CAPEC
scaffold was dipped in the dispersion slowly at the speed of 100mm/ minute for about 3 minutes.
Then the scaffold was withdrawn from the HAp suspension and allowed to dry. For one batch of
scaffolds, one more cycle of dip coating was performed. Scaffolds undergone only one cycle of
dip coating were named as CAPEC/HAp1, whereas the scaffolds with two cycles of dip coating
were designated as CAPEC/HAp2. The obtained CAPEC/HAp scaffolds were made ready for
different characterization techniques.
4.3 Characterization
4.3.1 Average particle size
HAp sample was subjected to Dynamic Light Scattering (DLS) in order to find out average
particle size. HAp powder was suspended in ethanol solution. The suspension was ultrasonicated
for 30 minutes in an ultrasonication bath, before analysis.
4.3.2 Morphological characterization
The morphology of developed scaffolds was observed by SEM (Scanning Electron Microscopy).
CAPEC scaffold and CAPEC/HAp scaffold samples were sputter-coated with Au/Pd and imaged
by SEM (SEM JEOL-JSM 6480 LV). Along with Scaffold samples, the morphology of
synthesized HAp samples was studied using SEM.
Page | 19
4.3.3 Porosity measurement
The overall porosity of the scaffold was measured using the fluid saturation method. A dry
sample of known weight (Wd) was taken. Its bulk volume (Vb) was calculated. The sample was
then immersed in water until it reaches saturation. Wet weight of the saturated scaffold was
calculated as Ww. Volume of water (Vc) in the pores was measured by dividing the difference
between wet weight and dry weight by density of water. The following equation was used to
measure the scaffold porosity. Samples were tested in duplicates and the average was taken and
calculated.
𝑃𝑜𝑟𝑜𝑠𝑖𝑡𝑦 % =𝑉𝑐
𝑉𝑏∗ 100
4.3.4 Phase analysis
Phase analysis of developed CAPEC scaffold , HAp powder and CAPEC/HAp composite
scaffolds was performed by X-ray Diffractometer [PANalytical, X’pertPhilips,USA] using Cu-
Kα radiation (λ= 0.1542 A°) at scanning rate of 100/ minute with step size of 0.05
0 within a
scanning region of 2θ = 10-500. The operating conditions were 30 kV and 30 mA.
4.3.5 Functional analysis
Fourier transform infrared spectroscopy (FTIR) was performed to identify organic and inorganic
compounds by determining the molecular composition and functional groups of the developed
CAPEC scaffold, HAp powder and CAPEC/HAp composite scaffold. An Infra red Microscope
[Shimadzu AIM-8800, Japan] was used for FTIR. The scaffold samples were pelletized using
Page | 20
hydraulic press by mixing them with dry KBr powder. The mixture was pressed into transparent
disks and used for IR analysis. The machine was operated in transmittance mode by using the
range 500 to 4000cm-1
with a resolution of 8cm-1
..
4.3.6 Mechanical strength
Prepared CAPEC & CAPEC/HAp scaffolds were tested for their mechanical strength
(compressive strength) using by Universal testing machine (H10 KS Tinius Olsen USA).
Scaffolds were cut in square shape with a length of 7 mm and thickness of 8 mm for analysis.
Compression test was performed with a crosshead speed of 1 mm/min with a load cell of 1kN
until 60% compression was achieved. Compressive strength was calculated by using following
formula;
𝑠 = (𝐹𝑚𝑎𝑥
𝐴)
Where, Fmax represents the force applied and A is the cross sectional area of the sample.
4.3.7 Swelling behavior
Swelling behavior of the developed CAPEC and CAPEC/Hap composite scaffolds was evaluated
using deionised water at room temperature until they reach equilibrium. The dry weight of the
scaffold was noted (WD). Scaffolds were then placed in deionized water for different time
intervals (1 h, 3 hrs, 5 hrs, 7 hrs, 24 hrs and 48 hrs). After each time interval, the scaffolds were
withdrawn and surface adsorbed water was removed using filter paper and the wet weight was
recorded (WT). The swelling ratio and water uptake % was calculated using following equations,
𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑟𝑎𝑡𝑖𝑜 =𝑊𝑇 − 𝑊𝐷
𝑊𝐷
Page | 21
𝑊𝑎𝑡𝑒𝑟 𝑢𝑝𝑡𝑎𝑘𝑒 % =𝑊𝑇 − 𝑊𝐷
𝑊𝐷∗ 100
4.3.8 In-vitro biodegradation study
The scaffolds of known dry weights (Wi) were sterilized by immersing in 70% ethanol before
starting the biodegradation study. Sterilized scaffold samples were then immersed in PBS (pH
7.4) at 37°C. The PBS solution was refreshed daily to ensure continuous degradation. The
soaking time of the samples was 1, 3, 5, 7, 14, 21 and 28 days. The samples were removed at
regular time intervals and vacuum dried before calculating the final weight, Wf. The extent of
degradation was expressed as a percentage of weight remained of the dried sample after
degradation. The percentage of weight remained was calculated using the following equation:
% 𝑤𝑒𝑖𝑔ℎ𝑡 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 = 100 – [𝑊𝑖 – 𝑊𝑓
𝑊𝑖∗ 100]
Page | 22
5. RESULTS & DISCUSSION
This study focuses on the development of CAPEC scaffold for the bone tissue engineering
application. The intention was to achieve desired pore size, porosity, mechanical strength,
swelling behavior, biodegradation rate. The prepared scaffolds were characterized for all these
required parameters. In order to improve the mechanical strength and bioactivity of the
developed CAPEC scaffold, they were coated with HAp. The HAp was synthesized by wet
chemical precipitation method. To confirm the synthesis and crystallinity of HAp,
characterization of prepared sample was done. Also, particle size of the synthesized HAp was
determined in order to confirm the nano-sized HAp formation. This chapter describes results and
discussion on this study.
5.1 Average particle size
Average particle size of synthesized HAp was studied using DLS. The average particle size of
HAp was found to be 159.2 nm. Figure 5.1.1 shows the size distribution of HAp by intensity.
Figure 5.1.1 Size distribution profile of HAp sample
Page | 23
5.2 Preparation of scaffolds
3D porous scaffolds from chitosan-alginate polyelectrolyte complex were prepared by freeze
drying technique. Using dip coating method, prepared scaffolds were coated with nano-HAp.
Scaffolds were designated as CAPEC (CAPEC cross-linked with CaCl2), CAPEC/HAp1 (one
cycle of coating) and CAPEC/HAp2 (two cycles of coating). The scaffolds developed are shown
in the following figure 5.2.1.
Figure 5.2.1 a) Developed CAPEC scaffold, b) CAPEC/HAp1 scaffold, c) CAPEC/HAp2
scaffold
5.3 Morphology & pore size
SEM analysis was performed on developed scaffolds. SEM images of developed scaffolds are
shown in the figure 5.3.1. CAPEC scaffold showed interconnected open pore structure. The
porous network is composed of both micro and micro pores, which is found to be optimum for
bone tissue engineering. The pore size ranged from ~30 to 280µm. SEM images of
CAPEC/HAp1 and CAPEC/HAp2 scaffold shows the presence of HAp on the walls of scaffold.
a b c
Page | 24
But the distribution of HAp was not uniform due to the presence of agglomerates of HAp. There
was no apparent change in the pore size of the scaffold.
Figure 5.3.1 SEM images of CAPEC scaffold (a & b), CAPEC/HAp1 scaffold (c & d) and
CAPEC/HAp2 scaffold (e & f)
5.4 Porosity
An ideal scaffold must have interconnected pores which facilitate the transport of cells, nutrients,
metabolites. % porosity of the developed CAPEC scaffold was found to be ~82% on an average.
Decrease in porosity was found in case of HAp coated CAPEC scaffolds. CAPEC/HAp1 and
CAPEC/HAp2 scaffold showed average porosity of ~76% and ~69%, respectively. This
descrease in porosity may be attributed to the deposition of HAp on the walls of scaffold.
a
d c
b
f e
Page | 25
However, the porosity of the CAPEC/HAP1 scaffold is still high enough for the bone tissue
engineering application.
5.5 Phase analysis
Phase changes, if any, in CAPEC scaffold due to crosslinking and HAp coating were analyzed
by XRD studies. Figure 5.5.1 shows X-ray diffraction patterns for CAPEC scaffold (before
crosslinking, CS-AG), CAPEC scaffold (after crosslinking), HAp, CAPEC/HAp1 scaffold. XRD
pattern of CS-AG shows sharp peaks corresponding to chitosan-alginate interaction. Whereas,
for crosslinked CAPEC scaffold, peaks are broader and the corresponding peak intensity is
decreased. For HAp powder, presence of sharp peaks at 2θ = 25.80, 31.8
0, 32.1
0, 32.9
0, 34.0
0
corresponds to its crystalline nature. For CAPEC/HAp scaffold, the peaks of chitosan-alginate
and HAp are present and show crystalline nature but the peaks are weaker and broader.
Figure 5.5.1 XRD spectra of CS-AG, CAPEC scaffold, HAp powder and CAPEC/HAp1
Scaffold
Page | 26
5.6 Functional analysis
Figure 5.6.1 shows the IR spectra of pure chitosan, pure alginate, HAp and CAPEC/HAp
Scaffold. IR spectra of pure chitosan shows the characteristic bands of amino group (1173cm-1
)
and amide group (1643cm-1). Alginate spectrum shows a peak at 1607 cm-1
specific for carbonyl
bond. In the IR spectra of chitosan and alginate, peaks at 1075 cm-1
and 1424 cm-1
corresponding
to carboxyl –COOH and C-O stretching can be seen. HAp spectrum shows band at 603 cm− 1
which signifies the vibration of hydroxyl ions, whereas bands at 1034 and 565 cm− 1
corresponds
to phosphate bending in HAp. In the spectra of CAPEC/HAp, peak shift of Amide group from
1643 to 1607 cm-1
is seen, while the amine group peak is absent. The shift in the amide group
peak and absence of amino group peak is may be due to the chitosan-alginate polyelectrolyte
interaction between the carboxyl and amine group of chitosan and alginate.
Figure 5.6.1 FTIR spectra of a) pure chitosan, b) pure alginate, c) CAPEC/HAp1 scaffold
and d) HAp powder.
Page | 27
5.7 Mechanical strength
Until the tissue is regenerated, a scaffold must provide mechanical integrity, it should act as a
support against the stress generated by new tissue formation. The developed scaffolds were
tested for their compressive strength. Figure 5.7.1 depicts the compressive strength of CAPEC
scaffold (Un-crosslinked, CS-AG), CAPEC scaffold (crosslinked), CAPEC/HAp1 scaffold and
CAPEC/HAp2 scaffold. After crosslinking with CaCl2, compressive strength of CAPEC scaffold
is increased from 0.23 MPa to 0.469 MPa. This increase in mechanical strength can be attributed
to the strong ionic interaction of Ca+2
with COO- of alginate chain[8]. Similarly, it is seen that
HAp coating also resulted in increase in compressive strength of the scaffold [27]. Compressive
strength for CAPEC/HAp1 and CAPEC/HAp2 scaffolds was found to be 0.61 MPa and 0.67
MPa, respectively. Increase in compressive strength can also be related to decrease in porosity
due to distribution of HAp along the walls of CAPEC scaffold.
Figure 5.7.1 Compressive strength (MPa) of CS-AG, CAPEC, CAPEC/HAp1 and
CAPEC/HAp2 Scaffolds
Page | 28
5.8 Swelling behavior
Figure 5.8.1 shows the shape retention of each scaffold sample after 2 days of incubation.
Swelling behavior of developed scaffolds; CAPEC, CAPEC/HAp1 and CAPEC/HAp2 scaffold
is shown in the following figure 5.8.2. It can be seen that the CAPEC scaffolds shows higher
swelling percentage (~450%) than the HAp coated CAPEC scaffolds (~330% and ~250%). It is
observed that the swelling % is decreased with the increase in HAp coating. The swelling
percentage for CAPEC/HAp1 scaffolds was found to be ~330%, whereas for CAPEC/HAp2
scaffolds it was found to be ~250%. This may be attributed to decrease in porosity as well as
decrease in the diffusion of water due to HAp depositions on the wall of scaffolds.
Figure 5.8.1 Images of CAPEC (a), CAPEC/HAp1 (b) and CAPEC/HAp2 (C) scaffolds after
two days of immersion in PBS.
a) CAPEC b) CAPEC/HAP1 c) CAPEC/Hap2
2 days 2 days 2 days
Page | 29
Figure 5.8.2 Percentage swelling of scaffolds as a function of sample immersion time in PBS:
CAPEC, CAPEC/HAp, CAPEC/HAp2 Scaffolds.
5.9 In-vitro biodegradation
For an ideal scaffold, rate of biodegradation must be equal to the rate of new tissue formation. In
order to achieve this, control of biodegradation rate of a scaffold is necessary. Developed
scaffolds were subjected to in-vitro biodegradation for a period of 28 days. Figure 5.9.1 show
the % loss in weight of the scaffold samples as a function of immersion time in PBS. It was seen
that the rate of degradation in case of HAp coated CAPEC scaffold was lower than that for the
CAPEC scaffold. Following figure shows the % weight remaining of the scaffold as a function of
time. CAPEC scaffold lost almost 32% of the weight during the period of study, whereas for
CAPEC/HAp1 and CAPEC/HAp2 scaffolds the % weight loss was ~30% and ~ 22%. Difference
in rate of biodegradation may correspond to increased mechanical stability due to HAp coating.
Page | 30
Figure 5.9.1 Percentage weight remaining of scaffolds as a function of sample immersion time
in PBS: CAPEC, CAPEC/HAp, CAPEC/HAp2 Scaffolds.
Page | 31
6. SUMMARY & CONCLUSION
In the present study, 3D porous scaffolds using chitosan-alginate polyelectrolyte complex were
developed by freeze drying method. The developed scaffolds were further modified by
employing dip coating with the HAp. The HAp was synthesized by wet chemical precipitation
method. Instead of using water as a solvent, ethanol was used as a solvent for dissolution of the
reactants. Average particle analysis of synthesized HAp shows the average particle size of HAp
as ~159.2 nm. XRD analysis of HAp also confirms the crystalline nature of synthesized HAp.
CAPEC scaffolds coated once with HAp were found to be optimum for non-load bearing bone
tissue engineering applications. SEM analysis revealed the similar morphology and pore size of
CAPEC and CAPEC/HAp scaffolds with interconnected pore network made up of both micro
and macropores (~44 to ~180µm). But the porosity was decreased with the increase in HAp
concentration. There was a decrease in porosity from ~82% to ~69% in case of CAPEC/HAp
scaffolds. But CAPEC/HAp1 scaffolds displayed ~76% porosity, which is high enough for tissue
engineering applications. Increase in the mechanical strength was observed in CAPEC scaffolds
after the coating with HAp. Compressive strength of CAPEC scaffolds was found to be 0.469
MPa, whereas in case of CAPEC/HAp1 and CAPEC/HAp2 scaffolds it was found to be
increased to 0.61 and 0.67MPa. The swelling behavior and in-vitro biodegradation studies also
suggest that the developed scaffolds are favorable. In conclusion, the developed CAPEC/HAp
can be used as a bone scaffold in case of soft bone. In future, the developed hybrid composite
scaffold might provide a promising alternative to bone grafts in bone tissue engineering.
Page | 32
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